Biochemistry and Ultrastructure of Connective Tissue

Collagen

has gly-X-Y amino acid structure, high content of proline and hydroxyproline

Structure – all collagens have triple alpha helix – three left-hand alpha-helix chains forming right-handed superhelix

N- and C-terminal ends of procollagen (precursor molecule) are not helical, and are cleaved off by peptidases
proline and lysine are hydroxylated post-transcriptionally; hydroxylysine is then glycosylated to form crossbridges

number of crossbridges determines tensile strength

staggered array of monomers forms banding pattern under electron microscopy and with certain dyes

Synthesis – specialized cells (e.g, fibroblasts) make protein chains – signal sequence controls assembly into triple helix

processed by Golgi and secreted

Function – each primary alpha helix has a molecule on the N-terminal end that determines its function

collagen types are simply different arrangements of these different helices; each has a different function in tissue

(1) fibrous collagens – I, II, III, V, XI – can be bundles (bone, skin) or fibers (tendon, cartilage)

collagen II is the primary collagen in cartilage – fibers parallel to the surface to resist sheer forces

(2) fibril-associated collagens with interrupting helices (FACIT) - IX, XII, XIV – cartilage

(3) network- or filament-forming collagens not associated with fibrils – IV, VI, VII, VIII, X, XIII – basement membr.

collagen X is found only at the growth plate of hypertrophic chondrocytes

Proteoglycans

form bulk of "ground substance" (non-collagenous material in ECM)

classified as secreted (into ECM), intracellular granule (e.g., S cells), membrane intercalated (e.g., heparin sulfate)

Structure – core protein (11-220 kD) with 1-100 glycosaminoglycan (linear disaccharide polymer) side-chains attached to it

can be large (aggrecan, versican) or small (decorin, biglycan, lumican, fibromodulin)
core proteins are diverse and often not related

Synthesis – protein synthesized inside cell; glycosaminoglycan chains added in Golgi (except hyaluronic acid)

glycosamine is synthesized from UDP-galNAc/UDP-gluNAc

hyaluronic acid is synthesized at the cell surface (by hyaluronan synthase) – too large to be packaged inside cell

Examples:

aggrecan – primary proteoglycan of hyaline cartilage

100 chondroitan sulfate chains attached to serines, also keratin sulfate
three globular domains – N-terminal globular domain binds link protein which stabilizes aggregation

aggregates wind between type II collagen to allow resistance of compression deformation (SO₄ repulsion) due to the large net negative charge of the many glycosaminoglycan chains that bind many water molecules

versican – family of large proteoglycans in skin, tendon, ligament – associated with type I collagen

fewer glycosamino chains, so forms more dense connective tissue

small leucine-rich proteoglycans (SLRPs) – leucine repeats cause horseshoe around collagen in three dimensions

decorin – many possible functions

regulates diameter of collagen fibers (knockout mouse has various sized collagen fibers and weak skin)

induces metalloproteases – may be involved in tissue remodeling – found in wound keloids/tumors

mediates inflammation – binds C1q, heparin cofactor II, and controls TGF-b

other SLRPs include lumican (in the cornea), fibromodulin, biglycan

Non-collaganous Proteins

Elastin – in elastic tissue – ropes (skin, elastic ligaments, lung), sheets (arteries), or honeycombs (elastic cartilage)

synthesized as tropoelastin subunits (soluble monomer) that rapidly polymerize to microfibrils
crosslinkage occurs via lysyl oxidase to form three-dimensional network – also contains much proteoglycan

Cell-interactive (adhesive) glycoproteins – have collagen- and cell-binding domains

fibronectin – binds cells to ECM – found in almost all connective tissue

tenascin – six branching hexabrachion held together with disulfide bonds

thrombospondin – in cartilage

laminin – binds collagen IV in basement membranes

Connective Tissue Matrix Homeostasis

bone is constantly turning over, cartilage hardly at all

arthritis – degradation overtakes synthesis – cartilage degraded to single molecules bound to hyaluronic acid

released to sinovial fluid Þ lymphatics/capillaries Þ kidney (urinary diagnosis?)

proteases – enzymes that break down ECM components – some specific, some less so

metalloproteinases – Zn bind and Ca⁺⁺ dependence – collagenase(collagen II), gelatinase, stromelysin(proteoglycans)

tissue inhibitors of metalloproteinases (TIMPs) naturally inhibit these molecules

serine proteases – activate metalloproteinases through plasmin-plasminogen activator system

include TPA, urokinase, elastase, kallikrine

cathepsin (includes cysteine proteases and aspartate proteases) – cleave proteoglycans and fibrillar collagens

aggrecanase – cleaves globular domains of aggrecan, implicated in osteoarthritis